Polymorphonuclear neutrophils and mast cells play major role in the host defense against bacterial, fungal and viral infections. Microorganisms or their products, can interact directly with these cells and induce the release of inflammatory mediators such as leukotrienes and histamine which have multiple effects essential to amplification and control of the inflammatory response. Leukotriene B4 is a potent chemotactic and chemokinetic factor which attracts neutrophils, eosinophils, monocytes and macrophages to sites of infection and tissue trauma. It promotes the aggregation of neutrophils, augments their adherence to endothelial cells, stimulates the release of lysozomal enzymes and the production of superoxide. It also inhibits the proliferation of lymphocytes via the induction of suppressor T cells and augments cytotoxic and natural killer cell activities. On the other hand, leukotrienes C4, D4, and E4 are the slow-reacting substances of anaphylaxis which cause long-lasting contraction of smooth muscle, bronchoconstriction, vasoconstriction, secretion of mucous, and an increase in vascular permeability. Histamine induces vasodilation, increase in vascular permeability, stimulation of secretory glands and contraction of smooth muscle. Furthermore, histamine modulates certain immune effector functions such as cell-mediated cytotoxicity, lymphocyte proliferation, lymphokine production, and immunoglobulin synthesis. In general, the biological actions of leukotriene B4 and histamine tend to potentiate the cellular immune response and stimulate the host defense against microbial infections.
It is widely recognized that the biological actions of leukotriene B4 relating to recruitment and stimulation of neutrophils are essential for the host defense against microbial infections. The migration of polymorphonuclear neutrophils from circulating blood to the focus of infection is one of the earliest events of the inflammatory process. The cells leave the circulation and are guided towards sites of infection and tissue trauma by chemotaxis, in response to chemotatic factors released in the vicinity of invading microorganisms. Leukotriene B4 is the most potent chemotactic stimulus for neutrophils. The chemotactic stimulus promotes cellular movement and enables a variety of subsequent steps essential to the host defense including attachment, engulfment, ingestion, degranulation, secretion of lysozomal enzymes into phagolysozomes, and generation of toxic oxygen radicals, which ultimately lead to killing of invading microorganisms. Neutrophils control the concentration of leukotriene B4 in their environment by generating and metabolizing this inflammatory mediator. Leukotriene B4 is bound by specific receptors on the cell surface, internalized, and enzymatically oxidized producing biologically inactive products, 20-OH-leukotriene B4 and 20-COOH-leukotriene B4. In the case of major injuries, such as severely burned patients, the chemotactic responsiveness of neutrophils is impaired. Their production and release of leukotriene B4 is diminished, the metabolism of leukotriene B4 to inactive 20-OH-leukotriene B4 and 20-COOH-leukotriene B4 is enhanced, and the expression of leukotriene B4 receptors on the cell surface is reduced. Decreased production of leukotriene B4 and reduced responsiveness to leukotriene B4 leads to inadequate recruitment of phagocytes at sites of tissue injury and precedes the onset of microbial invasion.
Lactoferrin is an iron-binding glycoprotein present in various biological fluids of mammals including milk, saliva, tears and mucous secretions, and released from activated polymorphonuclear neutrophils at sites of inflammation. Large quantities of bovine lactoferrin can be obtained by extracting this protein from raw skim milk or cheese whey originating from the milk of cows and, consequently, bovine lactoferrin is readily available as a commercial product of the dairy industry. In its iron-free state, lactoferrin exhibits broad-spectrum antimicrobial activity which is commonly attributed to its ability to chelate iron and produce an iron-deficient environment limiting microbial growth. The present inventors first discovered that peptides having more potent antimicrobial properties than lactoferrin are generated upon enzymatic digestion of this protein (Japanese Patent Application No.238364/90). The antimicrobial peptide derived from lactoferrin appears to function by a mechanism distinct from iron binding and is effective at low concentrations against various species of Gram-negative and Gram-positive bacteria, yeasts, and molds, including strains known to cause disease in humans and animals. The effect of this peptide against microorganisms is lethal causing a rapid loss of colony-forming ability. Considerable potential exists for the widespread commercial use of this peptide as a safe and effective antimicrobial agent and a novel process has been established for its large-scale manufacture as described in Japanese Patent Application No.150604/91.
It has been demonstrated that lactoferrin has activity to inhibit calcium ionophore-induced release of histamine from peritoneal mast cells [Theobald, K. et. al. (1987) Agents and Actions 20:10-16], however, the effect of lactoferrin on the release of other inflammatory mediators such as leukotrienes is unknown. Whether peptides derived from lactoferrin have activity to modulate the release of inflammatory mediators from cells of the immune system has not been studied previously.
The present inventors investigated for the first time the effects of peptides derived from lactoferrin on the release of inflammatory mediators from cells of the immune system. Surprisingly, they discovered that the antimicrobial peptide of lactoferrin described in Japanese Patent Application No.238364/90 is an immunostimulatory peptide having excellent activity to modulate the release of leukotriene B4 and histamine from polymorphonuclear neutrophils and mast cells, respectively, and thereby potentiate the cellular immune response. In contrast, lactoferrin does not exhibit such useful immunostimulatory activity.